N-alkylation of acridans

ABSTRACT

The present invention provides compounds used in the synthesis of chemiluminescent acridinium compounds and methods of producing these compounds. Specifically, methods are provided for the N-alkylation of acridan compounds using alkylating reagents. Typically, these alkylating reagents comprise a protected sulfonate group protected with an acid-labile protecting group.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.62/689,811 filed Jun. 25, 2018, which is incorporated by referenceherein in its entirety.

FIELD OF INVENTION

The present invention describes the synthesis of N-alkylated acridancompounds used in the synthesis of high light yield chemiluminescentacridinium compounds without using the carcinogenic chemical 1,3-propanesultone. The current process is also expected to be generally applicablefor the synthesis of other chemiluminescent acridinium compoundscontaining functionalized N-alkyl groups at the acridinium nitrogen.

BACKGROUND OF INVENTION

Chemiluminescent acridininum labels are widely used in automatedimmunoassays. These labels exhibit excellent chemiluminescent stabilitycompared to unsubstituted acridinium phenyl esters or acridiniumsulfonamides (Law et al, J. Biolumin. Chemilumin. 4 (1989): 88-98; U.S.Pat. No. 8,778,624). Acridinium labels containing N-sulfopropyl groupsare hydrophilic and display improved aqueous solubility compared to thecorresponding N-methyl analogs (Law et al.; U.S. Pat. No. 5,656,426).These chemiluminescent labels also have low non-specific binding whichcan be further alleviated by the incorporation of poly(ethylene)glycol(PEG) or zwitterions in the acridinium ester structure (Natrajan et al,Org. Biomol. Chem. 9 (2011): 5092-5103; Natrajan et al, Anal. Biochem.406 (2010): 204-213; U.S. Pat. No. 6,664,043).

The synthesis of acridinium labels containing N-sulfopropyl groups isordinarily accomplished by N-alkylation of the acridine precursors withthe reagent 1,3-propane sultone at high temperatures in neat reactionswhere the alkylating reagent is also the solvent (Law et al, U.S. Pat.No. 5,656,426). These harsh conditions for the N-alkylation reaction arenecessitated by the poor reactivity of the hindered acridine nitrogentowards alkylating reagents. Although the neat reaction can be used forpreparative purposes, a major disadvantage is that 1,3-propane sultoneis quite toxic and poses a significant health hazard (Bolt and Golka,Toxicol. Lett. 151 (2004): 251-254; Ulland et al, Nature 230 (1971):460-461). A synthetic protocol for the N-alkylation of acridinecompounds in ionic liquids with substantially reduced quantities of1,3-propane sultone compared to the neat alkylation reactions has alsobeen developed (Natrajan and Wen, Green Chem. 13 (2011): 913-921; U.S.Pat. No. 8,293,908). The increased reactivity of the acridine precursorswith 1,3-propane sultone in ionic liquids also enabled the synthesis ofa variety of functionalized acridinium compounds (e.g., acridiniumesters or acridinium sulfonamides) containing N-sulfopropyl groups withminimal side reactions or decomposition. Additionally, the synthesis ofunsubstituted acridinium compounds (i.e., wherein C2-C8 carbon positionsin the acridinium ring are each unsubstituted), can also be accomplishedby N-alkylation of the corresponding acridan with the reagent sodium3-bromopropane sulfonate in ionic liquids (U.S. Pat. No. 9,512,080;Natrajan and Wen, Green Chem. Lett. Rev. 6 (2013): 237-248). The acridanN-alkylation process eliminates the use of 1,3-propane sultone for thesynthesis of unsubstituted N-sulfopropyl acridinium esters.

Unfortunately, these processes are not suited for the synthesis ofelectron-rich acridinium compounds. For example, electron-rich acridanprecursors of these acridinium compounds are easily oxidized to theiracridine counterpart when heated with sodium 3-bromopropane sulfonate inionic liquids. As can be seen in synthetic schema (Si), theelectron-rich acridans undergo oxidation to acridine rather thanN-alkylation of the central ring nitrogen of the acridan system (i.e.,N-alkylacridan is not formed). In synthetic schema S1, the acridan iselectron-rich due to the presence of OR and R′ groups at the C-2 and C7positions of the acridan ring system.

It is therefore an object of the invention to provide methods ofproducing N-alkylated acridine compounds unencumbered by theselimitations.

SUMMARY

The present invention is partially premised on the discovery thatelectron-rich acridans (e.g., acridans containing one or two alkoxygroups at C-2 and/or C-7 of the acridan ring) can be alkylated withpowerful alkylating reagents such as trifluoromethanesulfonates(triflates). These electron-rich acridans are acridan compounds havingan electron density greater than the electron density of the sameacridan in which the carbons at each of the C(1)-C(8) carbons of theacridan ring are bound to hydrogen. For example, the electron-richacridan may have the structure of formula (A1):

wherein “m” and “n” are independently 0-4 (i.e., 0, 1, 2, 3, or 4) andat least one of “m” or “n” is greater than 0;R₁ and R₂ are independently selected from electron donating groups(e.g., alkoxy); andR₃ is hydrogen or a C₁₀-C₄₅ hydrocarbon radical (e.g., C₁-C₃₀, C₁-C₂₀,C₁-C₁₀, C₅-C₁₅, C₅-C₃₀, C₅-C₄₀, C₁₀-C₄₀) optionally substituted with oneor more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I) andwherein R₃ may optionally comprise a zwitterionic group (e.g. —Z) and/ora zwitterionic linker group (e.g., —Z^(L)—).

The reaction may proceed through nucleophilic attack by the nitrogenatom in the central ring of the acridan ring system on the carbon atomof R to which the triflate leaving group is bonded in the protectedsulfonate triflate reactant. In most embodiments, the hydrocarbonlinking group (—R_(L)—) of the protected sulfonate triflate comprises acarbon atom bound to the triflate leaving group. An example of such areaction with a conversion of ≥80% is illustrated by

The method for the N-alkylation of an acridan compound may comprisereacting the acridan compound with a protected sulfonate triflatecompound having the structure of formula (R1):

wherein G is an acid-labile protecting group; and—R_(L)— is independently selected at each occurrence from C₁₋₂₀ linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5, one, two, three, four, five) heteroatoms (e.g,O, S, N, P, F, Cl, Br, I), and combinations thereof, and wherein R_(L)optionally comprises a zwitterionic linker group (e.g., —Z^(L)—). Inmost embodiments, the acridan is N-alkylated at the central nitrogen ofthe acridan ring system.

These N-alkylation reactions, while slow, typically proceed in goodconversion (≥80%) and the products, N-alkylated acridans, may then beeasily oxidized to the N-alkyl acridinium compound (e.g., N-alkylacridinium esters, N-alkyl acridinium sulfonamides).

In some embodiments, methods for the synthesis of protected acridiniumcompounds are also provided comprising:

(a) N-alkylating an acridan with a protected sulfonate triflate; and

(b) oxidizing the N-alkylacridan to convert the N-alkylacridan to aprotected acridinium.

The synthesis of N-alkylacridans or N-alkylacridinium may furthercomprise the reduction of an acridine compound to produce the acridan.In some embodiments, the protected acridinium may be deprotected toproduce a zwitterionic acridinium in the presence of acid underconditions compatible with the reactants in such a deprotection.

These N-alkylation reactions using protected sulfonate triflates resultin the introduction of a sulfonate with a protecting group attached to ahydrocarbon to the central ring nitrogen of the acridan ring system.Subsequent oxidation of the N-alkylated acridan followed by removal ofthe protecting group on the sulfonate results in the formation of thealkoxy-substituted N-sulfopropyl acridinium compound. Without wishing tobe bound by theory, it is believed that the increased reactivity of theacridans compared to their acridine precursors combined with theincreased reactivity of the triflate alkylating reagent is mainlyresponsible for the increased conversion in this chemicaltransformation. Moreover, if the acridine analog is used for theN-alkylation reaction rather than the acridan, poor conversions aretypically observed. Such poor conversation occurs even with highlyreactive triflate alkylating reagents. For example, reaction of2,7-dimethoxyacridine methyl ester with 3-bromopropyltriflate led tovery poor conversion. Similarly, the use of a less reactive alkylatingreagent such as a bromide or even iodide with an electron-rich acridanalso led to poor conversion.

The N-alkylation of reactive, electron-rich acridans with a protectedsulfopropyltriflate leads to efficient N-alkylation yieldingN-alkylacridans containing sulfonate protecting groups. TheseN-alkylated acridans may be used in the synthesis of chemiluminescentacridinium compounds. In some embodiments, the N-alkylated acridan mayhave the structure according to formula (NA1):

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0;R₁ and R₂ are independently selected from electron donating groups;R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical optionally substitutedwith one or more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br,I) and wherein R₃ may optionally comprise a zwitterionic group (e.g. Z)and/or a zwitterionic linker group (e.g., —Z^(L)—);—R_(L)—, is independently selected at each occurrence from C₁₋₂₀ linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I,etc.), and combinations thereof, and wherein R_(L) optionally comprisesa zwitterionic linker group (e.g., —Z^(L)—); and G is an acid-labileprotecting group.

The syntheses described herein are particularly useful for N-alkylatingelectron-rich acridans. For example, the electron-rich acridan may berepresented by the structure of formula (A1):

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0;R₁ and R₂ are independently selected from electron donating groups; andR₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical (e.g., C₁-C₃₀, C₁-C₂₀,C₁-C₁₀, C₅-C₁₅, C₅-C₃₀, C₅-C₄₀, C₁₀-C₄₀) optionally substituted with oneor more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I) andwherein R₃ may optionally comprise a zwitterionic group (e.g. —Z) and/ora zwitterionic linker group (e.g., —Z^(L)—). These electron-richacridans are acridan compounds having an electron density greater thanthe electron density of the same acridan in which R₁ and R₂ are hydrogenat each occurrence

In certain embodiments, acridan is an electron-rich acridan containingalkoxy groups at C-2 and/or C-7 of the acridan ring and, the highlyreactive alkylating reagent is a trifluoromethanesulfonate. In preferredembodiments, the alkylating reagent is a sulfonate-protectedsulfopropyltriflate leading to an N-alkylated acridan containing anN-sulfopropyl group with a protecting group on the sulfonate. Oxidationof the acridan to the acridinium ester followed by removal of theprotecting group on the sulfonate leads to a protected acridiniumcompound. The protected acridinium compound may have the structure

This synthetic protocol completely eliminates the use of thecarcinogenic chemical 1,3-propane sultone for the assembly of high lightyield, electron-rich acridinium esters. The elimination of thecarcinogenic alkylating reagent 1,3-propane sultone provides a moreenvironmentally-friendly synthesis of these high light output labels forclinical diagnostics. Moreover, such reaction may or may not occur inthe presence of ionic liquids. The chemical synthesis described hereinutilize the reactivity of the acridan separate from the functional groupat the C-9 position of the acridinium ring (i.e., the carbon bound to R₃in formula (A1)). Typically, this functional group provideschemiluminescence in acridinium complexes by formations of excited stateacridone, which is the light emitting species. Since the functionalgroup at C-9 is not implicated in these N-alkylation schema, thesynthetic mechanisms described herein operate with a variety of acridancompounds including acridan esters and acridan sulfonamides. In someembodiments, the acridan is an acridan ester. In other embodiments, theacridan is an acridan sulfonamide. Accordingly, various classes ofchemiluminescent acridinium compounds (and their respectiveintermediates) may be produced by the methods described, includingchemiluminescent acridinium esters and chemiluminescent acridiniumsulfonamides.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is an HPLC trace of N-sulfopropyl isopropoxy acridinium ester(Compound 5) synthesized using the methods of the current invention.

FIG. 2 is an HPLC trace of N-sulfopropyl PEG acridinium ester (Compound6) synthesized using the methods of the current invention.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, includingthe examples and appended claims, are collected here. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure pertains.

Unless otherwise explicitly defined, the following terms and phrases areintended to have the following meanings throughout this disclosure:

All percentages given herein refer to the weight percentages of aparticular component relative to the entire composition, including thecarrier, unless otherwise indicated. It will be understood that the sumof all weight % of individual components within a composition will notexceed 100%.

The terms “a” or “an,” as used in herein means one or more. As usedherein, the term “consisting essentially of” is intended to limit theinvention to the specified materials or steps and those that do notmaterially affect the basic and novel characteristics of the claimedinvention, as understood from a reading of this specification.

The following definitions of various groups or substituents are used,unless otherwise described. Specific and general values listed below forradicals, substituents, and ranges, are for illustration only; they donot exclude other defined values or other values within defined rangesfor the radicals and substituents. Unless otherwise indicated, alkyl,alkenyl, alkynyl, alkoxy, and the like denote straight, branched, andcyclic groups, as well as any combination thereof.

The term “hydrocarbon” refers to a radical or group containing carbonand hydrogen atoms. Examples of hydrocarbon radicals include, withoutlimitation, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, andany combination thereof (e.g., alkyl-aryl-alkyl, etc.). As used herein,unless otherwise indicated, hydrocarbons may be monovalent ormultivalent (e.g., divalent, trivalent, etc) hydrocarbon radicals. Aradical of the form —(CH₂)_(n)—, including a methylene radical, i.e.,—CH₂—, is regarded as an alkyl radical if it does not have unsaturatedbonds between carbon atoms. Unless otherwise specified, all hydrocarbonradicals (including substituted and unsubstituted alkyl, alkenyl,alkynyl, aryl, aryl-alkyl, alkyl-aryl, etc.) may have from 1-45 carbonatoms (e.g., C₁-C₃₀, C₁-C₂₀, C₁-C₁₀, C₅-C₁₅, C₅-C₃₀, C₅-C₄₀, C₁₀-C₄₀,C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆,C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀,C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀, C₄₁, C₄₂, C₄₃, C₄₄,C₄₅). In certain embodiments, hydrocarbons will have from 5-35 or from1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms,including for example, embodiments having one, two, three, four, five,six, seven, eight, nine, or ten carbon atoms. For example, hydrocarbonsmay have from about 2 to about 70 atoms or from 4 to about 60 atoms orfrom 4 to about 20 atoms.

A “substituted” hydrocarbon may have as a substituent one or morehydrocarbon radicals, substituted hydrocarbon radicals, or may compriseone or more heteroatoms. Any hydrocarbon substituents disclosed hereinmay optionally include from 1-20 (e.g., 1-10, 1-5, etc.) heteroatoms.Examples of substituted hydrocarbon radicals include, withoutlimitation, heterocycles, such as heteroaryls. Unless otherwisespecified, a hydrocarbon substituted with one or more heteroatoms willcomprise from 1-20 heteroatoms. In other embodiments, a hydrocarbonsubstituted with one or more heteroatoms will comprise from 1-12 or from1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms.Examples of heteroatoms include, but are not limited to, oxygen,nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I), boron,silicon, etc. In some embodiments, heteroatoms will be selected from thegroup consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen(e.g., F, Cl, Br, I). In preferred embodiments, the heteroatoms may beselected from O, N, or S. In some embodiments, a heteroatom or group maysubstitute a carbon. In some embodiments, a heteroatom or group maysubstitute hydrogen. In some embodiments, a substituted hydrocarbon maycomprise one or more heteroatoms in the backbone or chain of themolecule (e.g., interposed between two carbon atoms, as in “oxa”). Insome embodiments, a substituted hydrocarbon may comprise one or moreheteroatoms pendant from the backbone or chain of the molecule (e.g.,covalently bound to a carbon atom in the chain or backbone, as in“oxo”).

In addition, the phrase “substituted with a[n],” as used herein, meansthe specified group may be substituted with one or more of any or all ofthe named substituents. For example, where a group, such as an alkyl orheteroaryl group, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

In some embodiments, any hydrocarbon or substituted hydrocarbondisclosed herein may be substituted with one or more (e.g., from 1-6 orfrom 1-4 or from 1-3 or one or two or three) substituents X^(sub), whereX^(sub) is independently selected at each occurrence from one or more(e.g., 1-20) heteroatoms or one or more (e.g., 1-10)heteroatom-containing groups, or X^(sub) is independently selected ateach occurrence from —F, —Cl, —Br, —I, —OH, —OR*, —NH₂, —NHR*, —N(R*)₂,—N(R*)₃*, —N(R*)—OH, —N(→O)(R*)₂, —O—N(R*)₂, —N(R*)—O—R*, —N(R*)—N(R*)₂,—C═N—R*, —N═C(R*)₂, —C═N—N(R*)₂, —C(═NR*)(—N(R*)₂), —C(H)(═N—OH), —SH,—SR*, —CN, —NC, —CHF₂, —CCl₃, —CF₂Cl, —CFCl₂, —C(═O)—R*, —CHO, —CO₂H,—C(O)CH₃, —CO₂, —CO₂R*, —C(═O)—S—R*, —O—(C═O)—H, —O(C═O)—R*,—S—C(═O)—R*, —(C═O)—NH₂, —C(═O)—N(R*)₂, —C(═O)—NHNH₂, —O—C(═O)—NHNH₂,—C(═S)—NH₂, —(C═S)—N(R*)₂, —N(R*)—CHO, —N(R*)—C(═O)—R*, —C(═NR)—OR*,—O—C(═NR*)—R*, —SCN, —NCS, —NSO, —SSR*, —N(R*)C(═O)—N(R*)₂, —CH₃,—CH₂—CH₃, —CH₂—CH₂—CH₃, —C(H)(CH₂)₂, —C(CH₃)₃, —N(R*)—C(═S)—N(R*)₂,—S(═O)₁₋₂—R*, —O—S(═O)₂—R*, —S(═O)₂—OR*, —N(R*)—S(═O)₂—R*,—S(═O)₂—N(R*)₂, —O—SO₃, —O—S(═O)₂—OR*, —O—S(═O)—R*, —O—S(═O)—R*,—S(═O)R*, —S(═O)—R*, —NO, —NO₂, —NO₃, —O—NO, —O—NO₂, —N₃, —N₂—R*,—N(C₂H₄), —Si(R*)₃, —CF₃, —O—CF₃, —O—CHF₂, —O—CH₃, —O—(CH₂)₁₋₆CH₃,—OC(H)(CH₂)₂—OC(CH₃)₃, —PR*₂, —O—P(═O)(OR*)₂, or —P(═O)(OR*)₂; where,independently at each occurrence, R* may be H or a C₁₋₁₀ or C₁₋₈ or C₁₋₆or C₁₋₄ unsubstituted hydrocarbon, including without limitation alkyl,alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g, benzyl),aryl-alkyl (e.g., toluyl). In some embodiments, X^(sub) may comprise aC₁-C₈ or C₁-C₆ or C₂-C₄ perfluoroalkyl. In some embodiments, X may be aC₁-C₅ or C₂-C₆ or C₃-C₅ heterocycle (e.g., heteroaryl radical). The term“halo” or “halogen” refers to any radical of fluorine, chlorine, bromineor iodine. In certain embodiments, X^(sub) is independently selected ateach occurrence from —OH, —SH, —NH₂, —N(R*)₂, —C(O)OR*, —C(O)NR*R*,—C(O)NR*R*, —C(O)OH, —C(O)NH₂, F, or —Cl. In some embodiments, X^(sub)is F. In some embodiments, R* is hydrogen, or lower alkyl (e.g., C₁-C₅linear or branched alkyl such as methyl, ethyl, propyl, or isopropyl).In some embodiments, R* is hydrogen, or lower alkoxy (e.g., C1-C5 linearor branched alkoxy such as methoxy, ethoxy, propoxy, or isopropoxy). Insome embodiments, X^(sub) is —CF₃ or —O—CF₃. In some embodiments,X^(sub) may provide an anionic charge to counterbalance any cationiccharge directly or indirectly covalently attached and in order to form azwitterion (e.g., in zwitterionic linking groups —Z^(L)— or zwitterionicgroups —Z). In some embodiments, X^(sub) may be carboxylate (—C(O)O⁻),sulfonate (—SO₃ ⁻), sulfate (—OSO₃ ⁻), phosphate (—OP(O)(OR^(P))O⁻), oroxide (—O⁻), and R^(P) is hydrogen or C₁₋₁₂ hydrocarbon optionallysubstituted with up to 10 heteroatoms.

It will be understood that the description of compounds herein islimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding with regard to valencies, etc., andto give compounds which are not inherently unstable. For example, anycarbon atom will be bonded to two, three, or four other atoms,consistent with the four valence electrons of carbon.

In general, and unless otherwise indicated, substituent (radical) prefixnames are derived from the parent hydride by either (i) replacing the“ane” or in the parent hydride with the suffixes “yl,” “diyl,” “triyl,”“tetrayl,” etc.; or (ii) replacing the “e” in the parent hydride withthe suffixes “yl,” “diyl,” “triyl,” “tetrayl,” etc. (here the atom(s)with the free valence, when specified, is (are) given numbers as low asis consistent with any established numbering of the parent hydride).Accepted contracted names, e.g., adamantyl, naphthyl, anthryl,phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, andtrivial names, e.g., vinyl, allyl, phenyl, and thienyl are also usedherein throughout. Radicals of steroids may also be designated with the“yl,” “diyl,” “triyl,” “tetrayl,” etc. suffixes. Conventionalnumbering/lettering systems are also adhered to for substituentnumbering and the nomenclature of fused, spiro, bicyclic, tricyclic,polycyclic rings.

The term “alkyl” refers to a saturated hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₆ alkyl indicates that the group may havefrom 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionallysubstituted, e.g., by one or more substituents. Examples of alkyl groupsinclude without limitation methyl, ethyl, i-propyl, isopropyl, andtert-butyl. Any alkyl group referenced herein (e.g., R, R′, R″, R₁, R₂,R₃, R₄, R₅) may have from 1-45 carbon atoms. In other embodiments, alkylgroups will have from 1-30 or from 1-20 or from 1-12 or from 1-8 or from1-6 or from 1-3 carbon atoms, including for example, embodiments havingone, two, three, four, five, six, seven, eight, nine, or ten carbonatoms.

The term “haloalkyl” refers to an alkyl group, in which at least onehydrogen atom is replaced by halo. In some embodiments, more than onehydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14,etc.) are replaced by halo. In these embodiments, the hydrogen atoms caneach be replaced by the same halogen (e.g., fluoro) or the hydrogenatoms can be replaced by a combination of different halogens (e.g.,fluoro and chloro). “Haloalkyl” also includes alkyl moieties in whichall hydrogens have been replaced by halo (sometimes referred to hereinas perhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Anyatom can be optionally substituted, e.g., by one or more substituents.

As referred to herein, the term “alkoxy” refers to a group of formula—O(alkyl). Alkoxy can be, for example, methoxy (—OCH₃), ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy,3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy” refers to agroup of formula —S(alkyl). Finally, the terms “haloalkoxy” and“halothioalkoxy” refer to —O(haloalkyl) and —S(haloalkyl), respectively.As used herein, the term “hydroxyl,” employed alone or in combinationwith other terms, refers to a group of formula —OH. Hydroxyalkyl refersto an alkyl group substituted with hydroxy (e.g., -(alky)-OH). Anyalkoxy, thioalkoxy, or haloalkoxy group referenced herein (e.g., R, R′,R″, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂) may have from1-35 carbon atoms. In other embodiments, alkyl, alkoxy, thioalkoxy, orhaloalkoxy groups will have from 1-30 or from 1-20 or from 1-12 or from1-8 or from 1-6 or from 1-3 carbon atoms, including for example,embodiments having one, two, three, four, five, six, seven, eight, nine,or ten carbon atoms.

The term “aralkyl” refers to an alkyl moiety in which an alkyl hydrogenatom is replaced by an aryl group (e.g., phenyl, naphthyl). One of thecarbons of the alkyl moiety serves as the point of attachment of thearalkyl group to another moiety. Any ring or chain atom can beoptionally substituted, e.g., by one or more substituents. Non-limitingexamples of “aralkyl” include benzyl, 2-phenylethyl, and 3-phenylpropylgroups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining the indicated number of carbon atoms and having one or morecarbon-carbon double bonds. Any atom can be optionally substituted,e.g., by one or more substituents. Alkenyl groups can include, e.g.,vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbonscan optionally be the point of attachment of the alkenyl substituent.Any alkenyl group referenced herein (e.g., R, R′, R″, R₁, R₂, R₃, R₄,R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂) may have from 1-35 carbon atoms. Inother embodiments, alkenyl groups will have from 1-20 or from 1-12 orfrom 1-8 or from 1-6 or from 1-3 carbon atoms, including for example,embodiments having one, two, three, four, five, six, seven, eight, nine,or ten carbon atoms.

The term “alkynyl” refers to a straight or branched hydrocarbon chaincontaining the indicated number of carbon atoms and having one or morecarbon-carbon triple bonds. Alkynyl groups can be optionallysubstituted, e.g., by one or more substituents. Alkynyl groups caninclude, e.g., ethynyl, propargyl, and 3-hexynyl. One of the triple bondcarbons can optionally be the point of attachment of the alkynylsubstituent.

The term “heterocyclyl” refers to a fully saturated, partiallysaturated, or aromatic monocyclic, bicyclic, tricyclic, or otherpolycyclic ring system having one or more constituent heteroatom ringatoms independently selected from O, N (it is understood that one or twoadditional groups (e.g., R^(N)) may be present to complete the tertiarynitrogen valence and/or form a salt unless otherwise indicated), or S.The heteroatom or ring carbon can be the point of attachment of theheterocyclyl substituent to another moiety. Any atom can be optionallysubstituted, e.g., with one or more substituents (e.g. heteroatoms orgroups X^(sub)). Heterocyclyl groups can include, e.g., tetrahydrofuryl,tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl(morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, thephrase “heterocyclic ring containing from 5-6 ring atoms, wherein from1-2 of the ring atoms is independently selected from N, NH, N(C₁-C₆alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heterocyclic ringis optionally substituted with from 1-3 independently selected R″ wouldinclude (but not be limited to) tetrahydrofuryl, tetrahydropyranyl,piperidyl (piperidino), piperazinyl, morpholinyl (morpholino),pyrrolinyl, and pyrrolidinyl.

The term “heterocycloalkenyl” refers to partially unsaturatedmonocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groupshaving one or more (e.g., 1-4) heteroatom ring atoms independentlyselected from O, N (it is understood that one or two additional groupsmay be present to complete the nitrogen valence and/or form a salt), orS. A ring carbon (e.g., saturated or unsaturated) or heteroatom can bethe point of attachment of the heterocycloalkenyl substituent. Any atomcan be optionally substituted, e.g., by one or more substituents.Heterocycloalkenyl groups can include, e.g., dihydropyridyl,tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl,4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro-pyrimidinyl, and5,6-dihydro-2H-[1,3]oxazinyl.

The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic,tricyclic, or other polycyclic hydrocarbon groups. Any atom can beoptionally substituted, e.g., by one or more substituents. A ring carbonserves as the point of attachment of a cycloalkyl group to anothermoiety. Cycloalkyl moieties can include, e.g., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl(bicycle[2.2.1]heptyl).

The term “cycloalkenyl” refers to partially unsaturated monocyclic,bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ringcarbon (e.g., saturated or unsaturated) is the point of attachment ofthe cycloalkenyl substituent. Any atom can be optionally substituted,e.g., by one or more substituents. Cycloalkenyl moieties can include,e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.

As used herein, the term “cycloalkylene” refers to a divalent monocycliccycloalkyl group having the indicated number of ring atoms.

As used herein, the term “heterocycloalkylene” refers to a divalentmonocyclic heterocyclyl group having the indicated number of ring atoms.

The term “aryl” refers to an aromatic monocyclic, bicyclic (2 fusedrings), or tricyclic (3 fused rings), or polycyclic (>3 fused rings)hydrocarbon ring system. One or more ring atoms can be optionallysubstituted, e.g., by one or more substituents. Aryl moieties include,e.g., phenyl and naphthyl.

The term “heteroaryl” refers to an aromatic monocyclic, bicyclic (2fused rings), tricyclic (3 fused rings), or polycyclic (>3 fused rings)hydrocarbon groups having one or more heteroatom ring atomsindependently selected from O, N (it is understood that one or twoadditional groups may be present to complete the nitrogen valence and/orform a salt), or S in the ring. One or more ring atoms can be optionallysubstituted, e.g., by one or more substituents. Examples of heteroarylgroups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl,4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, andxanthenyl.

In general, when a definition for a particular variable includes bothhydrogen and non-hydrogen (e.g., halo, alkyl, aryl) possibilities, theterm “substituent(s) other than hydrogen” refers collectively to thenon-hydrogen possibilities for that particular variable.

In general, the limits (end points) of any range recited herein arewithin the scope of the invention and should be understood to bedisclosed embodiments. For example, a range of 0 to 4 expresslydiscloses 0, 1, 2, 3, 4, and any subset within that range (e.g., from 0to 2, from 0 to 3, from 0 to 4, from 1 to 2, from 1 to 3, from 1 to 4,from 2 to 3, from 2 to 4, from 3 to 4).

The term “substituent” refers to a group “substituted” on, e.g., analkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group,replacing one or more hydrogen atoms therein. In one aspect, thesubstituent(s) on a group are independently any one single, or anycombination of two or more of the permissible atoms or groups of atomsdelineated for that substituent. In another aspect, a substituent mayitself be substituted with any one of the above substituents. Further,as used herein, the phrase “optionally substituted” means unsubstituted(e.g., substituted with an H) or substituted. It is understood thatsubstitution at a given atom is limited by valency. Common substituentsinclude halo (e.g. F), C₁₋₂ straight chain or branched chain alkyl,C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl, C₃₋₁₂ cycloalkyl, C₆₋₁₂ aryl, C₃₋₁₂heteroaryl, C₃₋₁₂ heterocyclyl, C₁₋₁₂ alkylsulfonyl, nitro, cyano,—COOR, —C(O)NRR′, —OR, —SR, —NRR′, and oxo, such as mono- or di- ortri-substitutions with moieties such as trifluoromethoxy, chlorine,bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl,piperazinyl, pyrazoyl, imidazoyl, and the like, each optionallycontaining one or more heteroatoms such as halo, N, O, S, and P. R andR′ are independently hydrogen, C₁₋₁₂ alkyl, C₁₋₁₂ haloalkyl, C₂₋₁₂alkenyl, C₂₋₁₂ alkynyl, C₃₋₁₂ cycloalkyl, C₄₋₂₄ cycloalkylalkyl, C₆₋₁₂aryl, C₇₋₂₄ aralkyl, C₃₋₁₂ heterocyclyl, C₃₋₂₄ heterocyclylalkyl, C₃₋₁₂heteroaryl, or C₄₋₂₄ heteroarylalkyl. Unless otherwise noted, all groupsdescribed herein optionally contain one or more common substituents, tothe extent permitted by valency. As used herein, the term “substituted”means that a hydrogen and/or carbon atom is removed and replaced by asubstituent (e.g., a common substituent). The use of a substituent(radical) prefix names such as alkyl without the modifier “optionallysubstituted” or “substituted” is understood to mean that the particularsubstituent is unsubstituted. However, the use of “haloalkyl” withoutthe modifier “optionally substituted” or “substituted” is stillunderstood to mean an alkyl group, in which at least one hydrogen atomis replaced by halo. Similarly, the use of “heteroalkyl” and other“hetero” modified hydrocarbons without the modifier “optionallysubstituted” is still understood to mean that a carbon atom in thehydrocarbon is replaced by O, N, or S.

In some embodiments the hydrocarbon (e.g., R, R′, R″, R₁, R₂, R₃, R₄,R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂) comprises a groups -L or —R_(L)-L,where L is a derivitizable functional group comprising a leaving group,electrophilic group, or nucleophilic group for forming a conjugate withan analyte, analyte analog, or binding partner for an analyte. In someembodiments, L will be selected from the group consisting of:

wherein R is independently at each occurrence hydrogen or a C₁-C₁₀hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aryl, arylalkyl); andR_(L) is a bivalent C₁-C₁₀ hydrocarbon (e.g., alkyl, alkenyl, alkynyl,aryl, arylalkyl).

In some embodiments, the hydrocarbon may comprise a zwitterionic group.The zwitterionic group may have the form:

wherein X^(L) is independently selected at each occurrence from a bond,—CH₂—, oxygen, sulfur, —NR^(N)—, amide (—NR^(N)(CO)—), carbamate(—NR^(N)C(O)O—), or urea (—NR^(N)C(O)NR^(N)—);R′ and R″ are independently selected at each occurrence from C₁₋₃₅alkyl, alkenyl, alkynyl, aryl, or aralkyl, each containing up to 20heteroatoms;Z₁ is a group —R_(L)—X^(a) where X^(a) is an anionic group such ascarboxylate (—COO⁻), sulfonate (—SO₃ ⁻), sulfate (—OSO₃ ⁻), phosphate(—OP(O)(OR^(P))(O⁻)), or oxide (—On is, independently selected at each occurrence, an integer between oneand 12;R^(P) is independently selected at each occurrence from C₁₋₃₅ alkyl,alkenyl, alkynyl, aryl, or aralkyl groups each containing up to 20heteroatoms;R_(L) may be absent (i.e., it is a bond) or a divalent radical selectedfrom C₁₋₃₁ alkyl, alkenyl, alkynyl, aryl, or aralkyl group, eachoptionally containing up to 20 heteroatoms; andR^(N) is lower alkyl (e.g., C₁-C₄ linear or branched alkyl such asmethyl, ethyl, propyl, isopropyl, butyl, isobutyl). More particularly, Zmay be a group:

where n is an integer from 1-12 and m is an integer from 0-3.

In some embodiments, a hydrocarbon may (e.g, —R_(L)—, R, R′, R″, R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂) comprise may comprise azwitterionic linker or a bivalent hydrocarbon linker with a zwitterioniclinking moiety. The zwitterionic linker may have the structure:

wherein “m” is 0 (i.e. it is a bond) or 1;

“n” and “p” are independently at each occurrence an integer from 0 (i.e.it is a bond) to 10;

X^(a) is an anionic group; and

R′ is hydrogen or lower alkyl (e.g., is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, etc.). Preferred zwitterioniclinkers have the formula:

The present invention provides compounds and methods for the synthesisof chemiluminescent acridinium compounds. Such compounds are able to beprepared without the carcinogenic reagent 1,3-propane sultone in thesynthesis of these commercially useful, high light yield, electron-richchemiluminescent acridinium labels containing N-sulfopropyl groups(e.g., acridinium dimethylphenyl esters, acridinium esters, acridiniumsulfonamides). Moreover, these synthetic protocols provide for thesynthesis of a variety of acridinium compounds with different functionalgroups attached to the central nitrogen of the acridinium ring system.

Exemplary high light yield, electron-rich acridinium esters capable offorming a conjugate with an analyte, analyte analog or other bindingpartner include acridinium esters 1, 2, 3, and 4. For acridinium esters1 and 2 that contain a C-2 isopropoxy group, compound 5 is the commonadvanced synthetic intermediate used for final assembly of theseacridinium esters. For the dialkoxy compounds 3 and 4, compounds 6 and 7represent the advanced synthetic intermediates respectively. The presentinvention includes compounds and methods useful for the production ofcompounds 5, 6, and 7 which are important precursors to Compounds 1-4.Compound 1-4 all display increased light output of 2-3 fold compared tounsubstituted acridinium esters (i.e., compounds without substitution atthe and C(2) and C(7) positions) and are useful labels for improvingsensitivity of immunoassays. Moreover, these production methods occurwithout the use of carcinogens like 1,3-propane sultone. Additionally,use of the reagents and protocols described herein allows for theattachment of sulfopropyl groups to the two ether oxygens of compound 7without the use of 1,3-propane sultone.

The acridans may be N-alkylated by reacting the acridan with a protectedsulfonate triflate compound having the structure of formula (R₁):

wherein G is an acid-labile protecting group; and—R_(L)— is independently selected at each occurrence from C₁₋₂₀ linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5, one, two, three, four, five) heteroatoms (e.g.,O, S, N, P, F, Cl, Br, I), and combinations thereof, and wherein R_(L)optionally comprises a zwitterionic linker group (e.g, —Z^(L)—). In someembodiments, the R^(L) of said sulfonate triflate compound is loweralkyl (e.g., C₁-C₄ linear or branched alkyl such as methyl, ethyl,propyl, butyl). In some embodiments, the sulfonate triflate compound hasthe structure of formula (R₂):

Typically, the protecting group for the sulfonate in the sulfonatetriflate undergoes cleavage from the sulfonate in acidic conditions.Such protecting groups have been described in Adamczyk et al, J. Org.Chem. 63 (1998): 5636-5639; Miller, J. Org. Chem. 75 (2010): 4632-4635;and Pauff and Miller, J. Org. Chem. 78 (2013): 711-716) each herebyincorporated by reference by their entirety and specifically in relationto sulfonate protecting groups and cleavage reactions. In someembodiments, G is selected from

In more preferred, embodiments, G is selected from

Typically, neopentyl protecting groups require strong acid for theirremoval. Additionally, synthesis of these groups requires using toxicoxirane and an organometallic reagent butyl lithium. (Adamczyk et al.).The α-trifluoromethylbenzyl (TFMB) sulfonates, TFMB and its 4′-methylversion (4′-Me-TFMB) are stable to mild acid and were described byMiller et al based on solvolysis studies of analogous compounds asreported by Allen et al, J. Am. Chem. Soc. 105 (1983): 2343-2354, eachhereby incorporated by reference by their entirety and specifically inrelation to sulfonate protecting groups and cleavage reactions. Theprotecting group may be cleaved following N-alkylation of the acridan.Preferably, the protecting group is cleaved from an acridinium compoundformed from the oxidation of the N-alkylated acridans.

Persons of ordinary skill are able to synthesize these protectedsulfonate triflates. For example, iodopropylsulfonate 4′-methyl TFMBester may be converted to the more reactive triflate by stirring theiodide with silver trifluoromethanesulfanate in tolune. In someembodiments, the invention provides the synthesis of protected sulfonatetriflates comprising reacting a compound having the structure:

with silver trifluoromethane sulfonate to produce a protected sulfonatetriflate having the structure of formula (R1):

wherein R_(L) is a bivalent hydrocarbon (e.g., propyl), andX^(sub) is halogen (e.g., —I). In certain embodiments G is:

Preferred alkylating reagents for the introduction of the N-sulfopropylgroup can be represented by the formula:

In most embodiments, G is selected from the following:

In preferred embodiments, G is 4′-Me-TFMB.

Typically, the cleavage reaction of the protected sulfonate protectinggroup occurs in an acid compatible with the acridan, acridine, oracridinium reactants. In some embodiments, the acid is a Brönsted acidwhich may be selected from hydrochloric acid, hydrobromic acid,sulphuric acid, benzenesulphonic acid, p-toluenesulphonic acid (p-TSA),methanesulphonic acid, ethanesulphonic acid, trifluoromethanesulfonicacid (TFMSA), trifluoroacetic acid (TFA), trichloroacetic acid (TCA),dichloroacetic acid (DCA), chloroacetic acid, formic acid and aceticacid. In some embodiments, the acid may be a Lewis acid or a siliconcompound or a combination of two or more such acids and/or siliconcompounds. The acid may be selected from boron trifluoride, borontrichloride, boron tribromide, aluminium chloride, tin chloride,titanium chloride, silicon tetrachloride, chlorotrimethylsilane Me₃SiCl(TMSCl), bromotrimethylsilane Me₃SiBr (TMSBr) and trimethylsilyltrifluoromethanesulphonate (TMSOTf). In preferred embodiments, the acidfor cleavage of the sulfonate protecting group may be trifluoroaceticacid (TFA), hydrochloric acid, or sulfuric acid etc.

The invention provides methods of N-alkylating acridans, andspecifically, electron-rich acridans. Accordingly, these methods allowfor the introduction of a hydrophilic, N-sulfopropyl group into a highlight yield, electron-rich chemiluminescent acridinium ester containingone or more alkoxy groups in the acridinium ring by (a) conversion ofthe acridine ester precursor to the corresponding acridan using areducing reagent; (b) N-alkylation of the acridan ester with asulfopropyl triflate where the sulfonate contains an acid-labileprotecting group by stirring the two components in a solvent under aninert atmosphere; (c) oxidation of the N-sulfopropylacridan with theprotected sulfonate to the N-sulfopropyl acridinium ester and; (d)cleavage of the sulfonate protecting group by acid hydrolysis. Theacridan may have the structure according to formula (A1):

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0;R₁ and R₂ are independently selected from electron donating groups; andR₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical (e.g., C₁-C₃₀, C₁-C₂₀,C₁-C₁₀, C₅-C₁₅, C₅-C₃₀, C₅-C₄₀, C₁₀-C₄₀) optionally substituted with oneor more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I) andwherein R₃ may optionally comprise a zwitterionic group (e.g. —Z) and/ora zwitterionic linker group (e.g., —Z^(L)—). In some embodiments, R₁and/or R₂ are alkoxy. In some embodiments, R₁ and R₂ are each the samealkoxy group (e.g. lower alkoxy such as methoxy, ethoxy, propoxy,isopropoxy, etc). In other embodiments, wherein R₁ or R₂ is hydrogen andthe other of R₁ or R₂ is alkoxy (e.g. lower alkoxy such as methyl,ethoxy, isopropoxy, isopropoxy, etc.). R1 and/or R2 may be independentlyselected from alkoxy groups having the structure:

wherein R_(a) is independently selected at each occurrence from methyl,isopropyl, or —(CH₂CH₂O)₁₋₁₀CH₃ (e.g., —(CH₂CH₂O) CH₃, etc.). In someembodiments the acridan has the structure of formula (A2):

In certain embodiments, the acridan has the structure of formula (A3):

wherein Ω is O or N;R₄ is absent when Ω is O or hydrogen or a C₁-C₄₀ (e.g., C₁-C₃₀, C₁-C₂₀,C₁-C₂₀, C₅-C₁₅, C₅-C₄₀) hydrocarbon radical optionally substituted withone or more (e.g., 1-15) heteroatoms (e.g, O, S, N, P, F, Cl, Br, I),thereof and wherein R₄ and R₅ may optionally comprise a zwitterionicgroup (e.g. —Z) and/or a zwitterionic linker group (e.g., —Z^(L)—); andR₅ hydrogen or a C₁-C₄₀ hydrocarbon radical optionally substituted withone or more (e.g., 1-15) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I),and combinations thereof and wherein R₄ and R₅ may optionally comprise azwitterionic group (e.g. —Z) and/or a zwitterionic linker group (e.g.,—Z^(L)—). The zwitterionic linker —Z^(L)— may have the structure:

wherein “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e.it is a bond) to 10;X^(a) is an anionic group; andR′ is hydrogen or lower alkyl (e.g., is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, etc.).

In preferred embodiments, acridan has the structure of formula (A4):

wherein R₆-R₁₀ are independently selected from hydrogen or a C₁-C₂₅(e.g., C₁-C₂₀, C₁-C₁₀, C₅-C₁₅, C₅-C₂₅) hydrocarbon radical (e.g., alkylsuch as methyl, ethyl, or propyl, alkoxy such as methoxy, ethoxy,propoxy, or isopropoxy) optionally substituted with one or more (e.g.,1-15) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I), and wherein R₆-R₁₀may optionally comprise a zwitterionic group (e.g. —Z) and/or azwitterionic linker group (e.g., —Z^(L)—). Typically, at least one ofR₆-R₁₀ is not hydrogen. In some embodiments, R₆ and R₇ are alkyl (e.g.,methyl). In some embodiments, at least one of R₆-R₁₀ comprises a leavinggroup for forming a conjugate with an analyte, analyte analog, orbinding partner for an analyte. In some embodiments, at least one ofR₆-R₁₀ is alkoxy. In preferred embodiments, the acridan has thestructure of formula (A5), (A6), or (A7):

wherein R₁₁ is alkyl, alkenyl, alkynyl, or aralkyl optionallysubstituted with one or more (e.g, 1-15) heteroatoms (e.g, O, S, N, P,F, Cl, Br, I).

The acridan may also have the structure of formula (A8):

wherein R₁₂ is a C₁-C₂₀ hydrocarbon (e.g., methyl, etc.).

In some embodiments, R₆-R₁₂ may comprise a leaving group for forming aconjugate with an analyte, analyte analog, or binding partner for ananalyte. For example, R₆-R₁₂ may comprise a group L selected from:

wherein R is independently at each occurrence hydrogen or a C₁-C₁₀hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aryl, arylalkyl); andR_(L) is a bivalent C₁-C₁₀ hydrocarbon (e.g., alkyl, alkenyl, alkynyl,aryl, arylalkyl). In preferred embodiments, R₆-R₁₂ are selected fromhydrogen, alkyl, —C(O)OH, —C(O)alkoxy, and alkoxy.

In some embodiments, the acridan may be produced by reducing thecorresponding acridine. For example an acridine having the structure

may be reduced to form an acidan having the structure of formula (A1).Similar corresponding acridines may be reduced to form acridans havingthe structure of formulas (A1)-(A8). In one embodiment, the acridineester has the formula:

wherein one of R₁ or R₂ is hydrogen or OR and the other of R₁ or R₂ isOR;R₆, R₇, and R₁₁ are R; andR is independently at each occurrence a C₁-C₄₀ (e.g., C₁-C₃₀, C₁-C₂₀,C₁-C₁₀, C₅-C₁₅, C₅-C₄₀) hydrocarbon radical (e.g., alkyl, alkenyl,alkynyl, aryl, aralkyl) optionally substituted with 1-20 heteroatoms(e.g., O, S, N, P, F, Cl, Br, I). In other embodiments, R₁ and R₂ areboth OR. In certain embodiments, R₆ and R₇ are methyl groups and R₁₁ isselected from methyl, ethyl, or isopropyl groups. In some embodiments,R₁ and R₂ are selected from lower alkoxy, and R₆, R₇ and R₁₁ are loweralkyl.

Preferred reducing reagents for the conversion of acridine to theacridan prior to N-alkylation are hydride reducing agents selected frompicoline-borane, sodium borohydride, lithium borohydride, potassiumborohydride, sodium cyanoborohydride or potassium cyanoborohydride.Alternatively, catalytic hydrogenation over a metal catalyst selectedfrom palladium or platinum on carbon can be used for the reduction.

The N-alkylated acridan may have the structure according to formula(NA1):

wherein R₁ and R₂ are independently selected from electron donatinggroups;R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical optionally substitutedwith one or more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br,I) and wherein R₃ may optionally comprise a zwitterionic group (e.g. —Z)and/or a zwitterionic linker group (e.g., —Z^(L)—);—R_(L)— is independently selected at each occurrence from C₁-20 linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I,etc.), and combinations thereof, and wherein R_(L) optionally comprisesa zwitterionic linker group (e.g., —Z^(L)—); andG is an acid-labile protecting group. These intermediate compounds areuseful in the synthesis of chemiluminescent acridinium compounds.Preferably, R_(L) is lower alkyl.

Following N-alkylation of the acridan, the N-alkylated acridan may bereduced to a protected N-alkylacridinium compound. In some embodiments,the protected N-alkylacridinium has the structure:

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0;R₁ and R₂ are independently selected from electron donating groups;R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical optionally substitutedwith one or more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br,I) and wherein R₃ may optionally comprise a zwitterionic group (e.g. —Z)and/or a zwitterionic linker group (e.g., —Z^(L)—);—R_(L)— is independently selected at each occurrence from C₁-20 linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5) heteroatoms (e.g., 0, S, N, P, F, Cl, Br, I,etc.), and combinations thereof, and wherein R_(L) optionally comprisesa zwitterionic linker group (e.g., —Z^(L)—); andG is an acid-labile protecting group.

An oxidation reaction of the N-alkylacridan may be used to produceacridinium compounds. In some embodiments, the oxidation occurs in anair environment. In other embodiment, oxidation of the N-alkylatedacridan may be achieved with molecular oxygen or DDQ(2,3-dichloro-5,6-dicyanobenzoquinone). In most embodiments, theprotected N-alkylacridinium may then unprotected using a cleavagereaction. However, in other embodiments, the sulfonate is deprotectedprior to oxidation of the N-alkylacridan. Typically, the cleavagereaction of the protected sulfonate protecting group occurs in an acidcompatible with the N-alkylacridinium reactants. In some embodiments,the acid is a Brönsted acid selected from hydrochloric acid, hydrobromicacid, sulphuric acid, benzenesulphonic acid, p-toluenesulphonic acid(p-TSA), methanesulphonic acid, ethanesulphonic acid,trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA),trichloroacetic acid (TCA), dichloroacetic acid (DCA), chloroaceticacid, formic acid and acetic acid. In some embodiments, the acid may bea Lewis acid or a silicon compound or a combination of two or more suchacids and/or silicon compounds. The acid may be selected from borontrifluoride, boron trichloride, boron tribromide, aluminium chloride,tin chloride, titanium chloride, silicon tetrachloride,chlorotrimethylsilane Me₃SiCl (TMSCl), bromotrimethylsilane Me₃SiBr(TMSBr) and trimethylsilyl trifluoromethanesulphonate (TMSOTf). In someembodiments, the acid for cleavage of the sulfonate protecting group maybe trifluoroacetic acid (TFA), hydrochloric acid, or sulfuric acid etc.In preferred embodiments, the acid is TFA.

Preferred solvents for any reaction (e.g., reduction, N-alkylation,oxidation, cleavage of protecting group or combinations thereof) arecommon organic solvents such as dichloromethane, chloroform,acetonitrile, toluene, etc. In some embodiments, the N-alkylation occursin anhydrous dichloromethane at room temperature under inert atmosphere(e.g., Argon).

Methods for the synthesis of N-alkylated acridiniums are provided whichmay comprise:

-   -   (a) reducing an acridine compound to convert said acridine        compound to an acridan:    -   (b) N-alkylating said acridan according to the method of any one        of claims 1-24 to produce an N-alkylacridan; and    -   (c) oxidizing said N-alkylacridan to convert said N-alkylacridan        to said acridinium.

In some embodiments, the method further comprises cleaving theacid-labile protecting group. In some embodiments, the acid-labileprotecting group is cleaved from the acridinium (i.e., to produce anunprotected N-alkylacridnium) by acid hydrolysis following saidN-alkylation step. In most embodiments, the cleaving may occur after theoxidizing step.

In some embodiments, the methods of the synthesis of chemiluminescentacridinium compounds comprises:

-   -   (a) reducing an acridine compound having the structure:

to produce an acridan having the structure:

(b) reacting said acridan with a first protected sodium triflatecompound having the structure:

to produce an N-alkylacridan having the structure:

and(c) oxidizing said N-alkylacridan to produce an N-alkylacridinium havingthe structure:

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0;R₁ and R₂ are independently selected from electron donating groups:R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radical optionally substitutedwith one or more (e.g., 1-20) heteroatoms (e.g., O, S, N, P, F, Cl, Br,I) and wherein R₃ may optionally comprise a zwitterionic group (e.g. —Z)and/or a zwitterionic linker group (e.g., —Z^(L)—);—R_(L)— is independently selected at each occurrence from C₁₋₂₀ linearor branched bivalent hydrocarbon radicals; optionally substituted withone or more (e.g., 1-5) heteroatoms (e.g., O, S, N, P, F, Cl, Br, I,etc.), and combinations thereof, and wherein R_(L) optionally comprisesa zwitterionic linker group (e.g., —Z^(L)—); andG is an acid-labile protecting group.

In some embodiments, the acridine has the structure

and said method further comprises reacting said acridine prior to saidreducing step with a second protected sulfonate triflate prior toacridine reduction having the structure

to produce an acridine having the structure

These methods may produce an N-alkylacridinium with the structure:

wherein the protecting group on the N-alkylated functional group may bethe same or different as the protecting group on the O-alkylatedfunctional groups.

In some embodiments, the acid-labile protecting groups may be removed toproduce an acridinium compound having the structure:

EXAMPLES

The following Examples illustrate the synthesis of a representativenumber of acridan and acridinium compounds. Accordingly, the Examplesare intended to illustrate but not to limit the disclosure. Additionalcompounds not specifically exemplified may be synthesized usingconventional methods in combination with the methods described herein.

The following examples describe the syntheses of compounds 5, 6 and 7using the methods of the present invention. These compounds are advancedprecursors to the acridinium esters 1-4.

Example 1: Synthesis of Compound 5

Reduction: A solution of 2-isopropoxyacridine methyl ester, compound i(Natrajan et al, Org. Biomol. Chem. 10 (2012): 3432-3447) (0.1 g, 0.225mmole) was dissolved in a mixture of tetrahydrofuran (THF, 9 mL) and 1 NHCl (1 mL). To this yellow solution was added solid picoline-boranecomplex (29 mg, 1.2 equivalents). The reaction was stirred at roomtemperature for 2 hours by which time the color of the reaction mixturehad faded to a light yellow color. HPLC analysis using a Phenomenexbondclone C₁₈, 3.9×30 mm column and a 30 minute gradient of 10→100%MeCN/water (each with 0.05% TFA) at a flow rate of 1 mL/minute and UVdetection at 260 and 220 nm showed product 2-isopropxyacridan methylester ii eluting at 22.8 minutes with very little starting material ieluting at 23.8 minutes. The reaction was concentrated under reducedpressure to remove the THF and the residual aqueous layer was dilutedwith ethyl acetate (25 mL) which was subsequently washed with 5% aqueoussodium bicarbonate solution followed by 5% aqueous NaCl. The ethylacetate solution was dried over anhydrous magnesium sulfate, filteredand concentrated under reduced pressure to give compound ii as a lightyellow solid. Yield=116 mg (quantitative). This material was used assuch for the N-alkylation reaction.

N-Alkylation: A solution of 2-isopropoxyacridan methyl ester (compoundii) (0.1 g, 0.225 mmole), triflate b (0.686 g, 0.00155 mole), and2,6-di-tert-butylpyridine (0.1 mL, 0.450 mmole) was stirred in anhydrousdichloromethane (2-3 mL) at room temperature under an argon atmosphere(balloon) protected from light for a week. TLC analysis (1:4, ethylacetate/hexanes) showed complete conversion to a less polar product.HPLC analysis as described above showed product eluting at 26.8 minutes.The reaction was concentrated under reduced pressure and the product iiiwas purified by flash chromatography on silica using ethylacetate/hexanes. Yield=107 mg (64%, sticky solid). MALDI-TOF MS 738observed.

The protected sulfopropyl iodide compound a was synthesized using aliterature procedure (Pauff and Miller, J. Org. Chem., 2013, 78,711-716). A solution of compound a (0.775 g, 0.00184 mole) in anhydroustoluene (10 mL) was treated with silver trifluoromethane sulfonate (0.47g, 0.00184 mole). The reaction was stirred at room temperature under anitrogen atmosphere protected from light. After 16 hours, a yellowprecipitate had formed in the reaction. The reaction was diluted withethyl acetate (25 mL) and was filtered. The filtrate was washed withcold water and then aqueous 5% NaCl (25 mL each). It was then dried overanhydrous magnesium sulfate and concentrated under reduced pressure togive a light brown oil which was used as such for the next reaction.Yield=0.686 g (compound b, 84%).

Oxidation: A solution of the N-alkylated acridan ester (compound iii)(66 mg, 0.089 mmole) in methanol (5 mL) was treated with DDQ (24.3 mg,0.0089 mmole). The reaction was stirred at room temperature. After 30minutes, HPLC analysis as described above showed complete conversion tothe acridinium ester eluting at 22 minutes. The solvent was removedunder reduced pressure.

Deprotection: The residue was stirred in TFA (1 mL) and water (0.02 mL)at room temperature. After 1 hour, HPLC analysis showed completeconversion to the product 5 eluting at 17.3 minutes. The reaction wasdiluted with methanol (5 mL) and concentrated under reduced pressure. Asimilar series of reactions where the oxidation was performed first oncompound iii followed by de-blocking of the sulfonate protecting groupto give crude 5 was also successful. The combined crude product 5 fromthese reactions (starting from 100 mg of 2-isopropxyacridan ii) waspurified by flash chromatography on silica using ethyl acetate/methanol.The product was isolated in excellent purity as illustrated in FIG. 1.Yield=37 mg (46% from iii, two steps).

Example 2: Synthesis of Compound 6

Reduction: The PEG acridine ester (compound v) precursor to acridan vihas been described previously (U.S. Pat. No. 7,309,615, herebyincorporated by reference in its entirety and specifically in relationto acridinium esters and their synthesis). Compound v (95 mg, 0.098mmole) was dissolved in THF (9 mL) and 1N HCl (1 mL). Picoline borane(21 mg, 2 equivalents) was added in one portion and the reaction wasstirred at room temperature. After 4 hours, the initial dark yellowsolution had faded to a light yellow color. HPLC analysis of thereaction as described previously showed acridan product vi eluting at18.7 minutes with very little starting material at 21 minutes. Thereaction was then concentrated under reduced pressure to remove the THFand the aqueous residue was partitioned between ethyl acetate (25 mL)and cold 1N HCl (25 mL). The ethyl acetate layer was separated and waswashed successively with aqueous 5% ammonium chloride, 5% sodiumbicarbonate and 5% NaCl. It was then dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The acridanvi was recovered as a white, sticky solid. Yield=90 mg (95%)

N-alkylation: A solution of acridan vi (90 mg, 0.092 mmole), triflate b(0.45 g, 1 mmole) and 2,6-di-tert-butylpyridine (0.04 mL, 2 equivalents)in dichloromethane (3 mL) was stirred under an inert atmosphere ofnitrogen (balloon), protected from light at room temperature for a week.HPLC analysis of the reaction mixture showed >90% conversion to theN-alkylated acridan vii eluting at 22.5 minutes. The reaction wasconcentrated under reduced pressure. The product was purified by flashchromatography on silica using a mixture of ethyl acetate and methanol.Yield=0.1 g (85%, sticky solid), MALDI TOF MS 1268 observed.

Oxidation: A solution of the N-alkylated acridan vii (0.1 g, 0.078mmole) in methanol (10 mL) was treated with DDQ (18 mg, 1 equivalent).The reaction was stirred at room temperature. After 30 minutes, HPLCanalysis showed complete oxidation to the acridinium ester eluting at20.2 minutes.

Deprotection: The solvent was then removed under reduced pressure andthe acridinium ester was stirred in TFA (1 mL) with water (0.02 mL) atroom temperature. After one hour, HPLC analysis showed >90% conversionto the product 6 eluting at 16.5 minutes. The reaction was treated withhexanes (30 mL) to precipitate the product. The hexanes were decantedand the residue was rinsed with hexanes (2×15 mL). The product was thendried under reduced pressure and then purified by flash chromatographyon silica using ethyl acetate/methanol. FIG. 2 shows the HPLC trace ofproduct 6 obtained following this synthesis. Yield=46 mg (50%). MALDITOF MS 1097 observed.

Example 3: Synthesis of Compound 7

A mixture of compound viii (U.S. Pat. No. 7,309,615, hereby incorporatedby reference in its entirety and specifically in relation to acridiniumesters and synthesis thereof) (50 mg, 0.12 mmole), potassium carbonateanhydrous (36 mg, 2.2 equivalents), 18-crown-6(1,4,7,10,13,16-hexaoxacyclooctadecane) (70 mg, 2.2 equivalents) wastreated with a MeCN solution (10 mL) of iodide a (127 mg, 2.5equivalents). The reaction was heated at 85° C. in an oil bath. Theinitial dark red solution faded away to a light brown color after 3hours. HPLC analysis as described previously showed the formation of amajor product eluting at 28 minutes. The reaction was cooled to roomtemperature and concentrated under reduced pressure. A similar scalereaction but without the addition of crown ether afforded a similarreaction profile but product formation required heating for 6 hours. Thecrude product from both reactions were combined and purified by flashchromatography on silica using a mixture of ethyl acetate/hexanes.Yield=0.1 g (42%). MALDI TOF MS 1006.4 observed.

Reduction: A solution of acridine ester ix (0.1 g, 0.096 mmole) in THF(18 mL) and 1N HCl (2 mL) was treated with solid picoline borane (21.3mg, 2 equivalents). The reaction was stirred at room temperature for 12hours. HPLC analysis showed acridan x eluting at 26.3 minutes with verylittle starting material at 28 minutes. The THF was removed underreduced pressure and the residue was partitioned between cold 1N HCl (25mL) and ethyl acetate (50 mL). The ethyl acetate extract was washedsuccessively with 25 mL each of aqueous 5% ammonium chloride, 5% sodiumbicarbonate and 5% sodium chloride. It was then dried over anhydrousmagnesium sulfate, filtered and concentrated under reduced pressure.Crude Yield=0.13 g. This material was used such for the N-alkylationreaction.

N-alkylation: The acridan compound x (0.13 g crude, 0.1 mmole) wasdissolved in anhydrous dichloromethane (4 mL) and treated with triflateb (0.43 g, 0.97 mmole) and 2,6-di-tert-butylpyridine (0.057 mL, 2.5equivalents). The reaction was stirred under an inert nitrogenatmosphere at room temperature for a week. HPLC analysis showed ˜80%conversion to product xi eluting at 28.3 minutes. (A similar reactionperformed on a 44 mg scale of acridan x showed complete conversion toproduct after two weeks.) The reaction was concentrated under reducedpressure and the product was purified by flash chromatography on silicausing a mixture of ethyl acetate and hexanes. Yield=73 mg (56%). MALDITOF MS 1300 observed.

Oxidation: A solution of the N-alkylated acridan xi (73 mg, 0.056 mmole)in 1:1 methanol/ethyl acetate (10 mL) was treated with DDQ (14 mg, 1.1equivalents). The reaction was stirred at room temperature. After 10minutes, HPLC analysis showed complete conversion to the acridiniumester eluting at 25 min.

Deprotection: The solvent was removed under reduced pressure and theresidue was stirred in TFA (2 mL) and water (0.04 mL). After 1.5 hours,HPLC analysis showed a major product compound 6 eluting at 11.3 minutes.The solvent was removed under reduced pressure and the residue wasrinsed with ether (50 mL) followed by 1:1 ether/hexanes and then 1:1hexanes/ethyl acetate. The crude product was then dried under reducedpressure. Crude Yield=60 mg. MALDI TOF MS 784 observed.

All references including patent applications and publications citedherein are incorporated herein by reference and for all purposes to thesame extent as if each individual publication or patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes. Manymodifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

The invention claimed is:
 1. A method for the N-alkylation of an acridancompound comprising reacting said acridan compound with a protectedsulfonate triflate compound having the structure of formula (R1):

wherein G is an acid-labile protecting group; and —R_(L)— isindependently selected at each occurrence from C₁₋₂₀ linear or branchedbivalent hydrocarbon radicals; optionally substituted with one or moreheteroatoms, and combinations thereof, and wherein R_(L) optionallycomprises a zwitterionic linker group.
 2. The method according to claim1, wherein R^(L) of said sulfonate triflate compound is lower alkyl. 3.The method according to claim 1, wherein G is selected from


4. A method for synthesis of an acridinium comprising: (a) reducing anacridine compound having the structure:

to produce an acridan having the structure:

(b) reacting said acridan with a first protected sodium triflatecompound having the structure:

to produce an N-alkylacridan having the structure:

and (c) oxidizing said N-alkylacridan to produce an N-alkylacridiniumhaving the structure:

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0; R₁ and R₂ are independently selected from electrondonating groups; R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radicaloptionally substituted with one or more heteroatoms and wherein R₃ mayoptionally comprise a zwitterionic group and/or a zwitterionic linkergroup; —R_(L)— is independently selected at each occurrence from C₁₋₂₀linear or branched bivalent hydrocarbon radicals; optionally substitutedwith one or more heteroatoms, and combinations thereof, and whereinR_(L) optionally comprises a zwitterionic linker group; and G is anacid-labile protecting group.
 5. The method according to claim 4 furthercomprising removing said acid labile protecting group to produce acompound having the structure:


6. The method according to claim 4, wherein said acridine iselectron-rich.
 7. The method according to claim 4, wherein said acridinehas the structure:


8. An N-alkylated acridan compound having the structure according toformula (NA1):

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0; R₁ and R₂ are independently selected from electrondonating groups; R₃ is hydrogen or a C₁-C₄₅ hydrocarbon radicaloptionally substituted with one or more heteroatoms and wherein R₃ mayoptionally comprise a zwitterionic group and/or a zwitterionic linkergroup; —R_(L)— is independently selected at each occurrence from C₁₋₂₀linear or branched bivalent hydrocarbon radicals; optionally substitutedwith one or more heteroatoms, and combinations thereof, and whereinR_(L) optionally comprises a zwitterionic linker group; and G is anacid-labile protecting group.
 9. The N-alkylated acridan according toclaim 8, wherein —R_(L)— is lower alky.
 10. An N-alkylated acridiniumcompound having the structure according to formula (NA2):

wherein “m” and “n” are independently 0-4 and at least one of “m” or “n”is greater than 0; R₁ and R₂ are independently selected from electrondonating groups; R₃ is hydrogen or a C1-C45 hydrocarbon radicaloptionally substituted with one or more heteroatoms and wherein R₃ mayoptionally comprise a zwitterionic group and/or a zwitterionic linkergroup; —R_(L)— is independently selected at each occurrence from C₁₋₂₀linear or branched bivalent hydrocarbon radicals; optionally substitutedwith one or more heteroatoms, and combinations thereof, and whereinR_(L) optionally comprises a zwitterionic linker group; and G is anacid-labile protecting group.
 11. The N-alkylated acridan according toclaim 8, wherein R_(L) is lower alkyl.